Many human blood proteins are in short or limited supply due to the larger quantities required of the protein for positive therapeutic effect or possibly also due to the larger demand of these proteins by the world population of patients having the particular condition. It is also advantageous to produce blood proteins, normally extracted from blood products, from an alternative source such as crop plants. Production of blood proteins from plants mitigates contamination of the blood protein fraction with human viruses and other disease causative agents found in human or animal blood product fractions.
Blood proteins such as hemoglobin, alpha-1-antitrypsin (“AAT”), fibrinogen, human serum albumin, thrombin, antithrombin III, antibodies, blood coagulation factors (e.g. Factors V-XIII), and others are known to have therapeutic potential for a number of human conditions.
Hemoglobin is the major blood component molecule transporting oxygen to cells. Mammalian hemoglobins are tetrameric proteins made up of two ex-like polypeptide subunits and two non-α (usually β, γ, or δ) subunits. These subunits differ in primary amino acid sequence, but have similar secondary and tertiary structures. Each globin subunit has associated with it, by noncovalent interaction, a Fe2+-porphyrin complex known as a heme group, to which oxygen binds. The predominant hemoglobin in adult erythrocytes is α2β2, known as hemoglobin A1 (HbA). Each hemoglobin tetramer has a molecular weight of 64 kD and each α-like and β-like chain has a molecular weight of approximately 15.7 kD (141 amino acids) and 16.5 kD (146 amino acids) respectively.
AAT belongs to the class of serpin inhibitors and is one of the major protease inhibitors in human plasma. AAT is a single 394 amino acid polypeptide having an approximate molecular mass of 52 kD, and contains about 15% carbohydrate in the native human form of the molecule. Concentrations of AAT in human plasma range from 1000-3000 mg/L and in human milk range from 100 to 400 mg/L Its primary physiological role is the inhibition neutrophil elastase, with an insufficiency leading to the development of pulmonary emphysema. Excess production of elastase activity leads to emphysema, hepatitis and a variety of skin disorders. While the binding affinity of AAT is highest for human neutrophil elastase, it also has affinity for pancreatic proteases such as chymotrypsin and trypsin. The current primary source for the treatment of AAT deficiency is isolating AAT from human blood plasma.
Fibrinogen is involved in the blood coagulation cascade and is converted to fibrin by its interaction with the natural clotting agent thrombin. Fibrin is the major component of blood clotting. Mature human fibrinogen consists of two pairs of three independent polypeptide chains (α, β and γ) that are linked together by 29 intra- and intermolecular disulfide bonds forming a native protein of 340 kD and is present in human plasma at an approximate concentration of 2500 mg/L. Three-dimensional structural analysis of independent fibrinogen domains has provided detailed structural features giving important clues to human fibrinogen's multifunctional role. The fibrinogen polypeptides are approximately 72 kD (α), 52 kD (β) and 48 kD (γ) respectively with the β polypeptide chain determining native molecule assembly. The structure of fibrinogen features a number of structural and functional domains containing multiple binding sites that facilitate interactions with itself, other proteins, certain cell types and allow fibrinogen to participate in a number of important physiological processes including blood coagulation, inflammation, angiogenesis, wound closure, arteriogenesis and tumorigenesis. Fibrin formation from a clotting standpoint is mediated by the interaction of native fibrinogen with its natural clotting agents Factor XIII and thrombin in the presence of blood soluble calcium.
Albumin is a transport protein molecule that carries out many functions in mammalian serum biology, notably that of a carrier of hormones and other soluble ligands from site to site, and other activities that contribute largely to general mammalian biochemistry. Human serum albumin (“HSA”) is also the major protein component of blood being actively present at serum concentrations of approximately 30,000-50,000 mg/L. HSA is a single polypeptide chain of 66.5 kD that is initially synthesized as a prepro-albumin molecule in the liver and released from the endoplasmic reticulum after N-terminal and C-terminal Golgi processing. The resultant mature protein is 585 amino acids in length. It has been shown that the natural preprosequence of HSA can function in correct protein targeting/processing across a plant plasma membrane in transgenic tobacco leaves (Sijmons et al., 1990).
Prothrombin, a plasma glycoprotein, is the zymogen of the serine protease thrombin that catalyzes the conversion of fibrinogen to fibrin as well as several other reactions that may be important for blood coagulation. Prothrombin is a single polypeptide chain approximately 72,000 molecular weight in size. The complete human thrombin cDNA consists of 622 amino acid residues and includes a leader sequence of 36 amino acid residues. Active thrombin has an apparent molecular weight of 36,000 and is made up of two disulfide-linked polypeptide chains resulting from prothrombin cleavage. The proteolytic events leading to in vitro activation and conversion of human prothrombin to active thrombin have been extremely well characterized. Antithrombin III is a single chain glycoprotein with a molecular weight of 58 kD. It is a member of the serpin (serine protease inhibitor) super family and is considered to be the most important inhibitor in the coagulation cascade. Antithrombin III inhibits a wide spectrum of serine proteases including thrombin, factors IXa, Xa and Xla, kallikrein, plasmin, urokinase, C1-esterase, and trypsin. Antithrombin III activity is markedly potentiated by heparin; potentiation of its activity is the principle mechanism by which both heparin and low-molecular-weight heparin produce anticoagulation.
Factors V-XIII are proteins (mostly proteases in their active states) that are involved in the ‘intrinsic pathway’ of the classical casade mechanism for blood coagulation. The majority of these molecules exist as precursors that are processed in an ordered sequence of transformations from inactive to catalytically active forms. Factor V is proaccelerin (the accelerator globulin) while Factor VI is the activated form of Factor V. Factor VII is proconvertin, the plasma thromboplastin component, while Factors VIII (antihemophilic factor) and IX (Christmas antihemophilic factor) are both associated with the hemophilia disease state. Factors X (Stuart-Power factor), XI (plasma thromboplastin anticedent) and XII (Hageman factor) are all involved with the maturation/stabilization of thrombin. Factor XIII (fibrin stabilizing factor) is a plasma transglutaminase directly acting on fibrin during the clotting process. All these Factors are present at relatively low in serum plasma (0.001 to 50 mg/L). Other protein factors also involved in the blood coagulation cascade include Fletcher Factor (prekallikrein), Fitzgerald factor (kininogen) and von Willebrand Factor.
Immunoglobulins (antibodies) present in humans act to confer resistance to a variety of pathogens to which a patient may have been exposed. Immunoglobulin molecules account for 15-20% of the mass in human serum and consist predominantly of IgG, IgM and IgA-type antibodies involved in fighting various infections that invade the blood system and potentially the rest of the body. IgG type antibodies are the most prevalant and exist at a serum concentration of between 618 g/L. The blood system also serves as a carrier directing these molecules to specific areas of the body to combat resulting infections and potential oncogenic targets. Mature antibodies consist of two polypeptides (light and heavy chains) that must be expressed in eqimolar amounts and come together to form functional entities. The light chain (˜25 kD) is a protein of ˜210-240 amino acids in length while the heavy chain (˜50 kD) is a protein of ˜450-460 amino acids in length. Both light and heavy chains carry signal peptides for processing and secretion into the blood stream. Expression of monoclonal antibodies in plants is of particular interest, because it requires the expression of two genes, synthesis of two proteins and correct assembly of the tetrameric protein to result in a functional antibody.
Initial studies of antibodies in plants focused on the IgG antibody class (Hiatt et al., 1989; Hiatt and Ma, 1992), but later studies explored the in planta expression of complex antibody molecules such as secretory IgA antibodies (4 genes) and more complex antibody forms (Ma et al., 1995; Vine et al., 2001).
U.S. Pat. Nos. 6,471,429, 5,959,177, 5,639,947 and 5,202,422, all related patents, disclose the production of antibody molecules in transgenic tobacco plant leaves.
U.S. Pat. No. 6,303,341 discloses the production of immunoglobulins containing protection proteins in tobacco plant leaves, stems, flowers and roots.
Published U.S. Patent Application U.S. 2002/0174453 discloses the production of antibodies in the plastids of tobacco plants.
Published U.S. Patent Application U.S. 2002/0046418 discloses a controlled environment agriculture bioreactor for the commercial production of heterologous proteins in transgenic plants. The specification discloses that production of mammalian blood proteins can be achieved. Example 7 discloses the production of human blood factors in the leaves of potato, tobacco and alfalfa plants.
U.S. Pat. No. 6,344,600 discloses the production of hemoglobin and myoglobin in tobacco plant leaves. Example X discloses the extraction and partial purification of recombinant hemoglobin from tobacco seeds. The expression was obtained by transformation of the coexpression plasmid pBIOC59, which was constructed to allow targeting in the chloroplasts, and contained for this purpose the transit peptide of the precursor of the small subunit of ribulose 1,5-diphosphate carboxylase of Pisum sativum L. Expression in seeds was reported to be at a maximum level of 0.05% recombinant hemoglobin relative to the total soluble proteins extracted.
Example XI of the '600 patent discloses the construction of plasmids containing one of the α or β chains of hemoglobin allowing constitutive expression or expression in the albumin in maize seeds. According to this disclosure, the constitutive or albumin-specific expression of the hemoglobin chains required the following regulatory sequences: one of three promoters allowing a constitutive expression ((i) the rice actin promoter followed by the rice actin intron, contained in the plasmid pAct1-F4; (ii) the 35S double constitutive promoter of cauliflower mosaic virus; or (iii) the promoter of the maize γ-zein gene contained in the plasmid pγ63) and one of two terminators ((i) the 35S polyA terminator; or (ii) the NOS polyA terminator). No experiment or data is provided regarding transformation or expression of these plasmids in maize or maize seeds.
U.S. Pat. No. 5,767,363 discloses the use of a seed-specific promoter derived from ACP of Brassica napus, to affect and vary the expression of seed oils in rape and tobacco plants. The specification generically discloses that the seed specific promoter can be used for the expression of pharmaceutical proteins, such as blood factors or human serum albumin, however no experimental data whatsoever is presented in this regard.
Daniell et al. (2001) is a review article discussing recent developments in the field of medical molecular farming, including the production of antibodies and proteins in plants.
None of these patents or publications discloses the production of human blood proteins in monocot seeds in high yield. It is desirable to provide for the production of human blood proteins in high yield free from contaminating source agents in order to provide the patient population with sufficient supply of these proteins for use in treating humans with conditions treatable by administration of a particular blood protein.